KR100498912B1 - Peptide for Binding to psi RNA Sequence of HIV - Google Patents

Abstract

The present invention relates to a peptide that binds specifically to the psi RNA sequence of HIV and a gene encoding the electrical peptide. A peptide capable of inhibiting the proliferation of HIV-1 by binding to the psi sequence of the present invention is a peptide having an amino acid sequence of SYQWWWHSPQTL. The peptide of the present invention binds to the psi RNA sequence, inhibits the binding between the psi RNA sequence and the NC protein, thereby inhibiting the proliferation of HIV, and thus may be widely used for the treatment of effective acquired immunodeficiency syndrome.

Description

Peptide for Binding to psi RNA Sequence of HIV}

The present invention relates to a peptide that specifically binds to the psi RNA sequence of human immunodeficiency virus (HIV). More specifically, the present invention relates to peptides that specifically bind to psi RNA sequences of HIV and genes that encode electrical peptides.

According to the World Health Organization (WHO), 34 million people worldwide are infected with the AIDS virus, and the number is increasing by 20-30% annually. The AIDS infected and infected population is expected to reach 300 million in 2005. Approximately 13.9 million people have died from AIDS since the AIDS epidemiological survey began. Human Immunodeficiency Virus (HIV) is a type of retrovirus that causes immunosuppressive diseases related to mammalian hematopoietic and central nervous systems and is classified as lentivirus. HIV is transmitted to others through semen and blood, and it damages the body's immune system, reducing its ability to fight pathogens. It takes a very long time (on average 8-10 years) to develop, and in 1986 A new form was discovered, the former being named HIV-1 and the new HIV-2. Of these, HIV-1 (Human Immunodeficiency Virus Type 1) is a retrovirus and is a positive sense single strand RNA virus. The gene for HIV-1 has nine ORFs, three of which are Gag, Pol, and Env, and the other six are responsible for regulatory functions. In particular, Gag and Env are structural components, Gag expresses four proteins of MA (matrix), CA (capsid), NC (nucleocapsid) and p6, and Env is SU (surface or gp120) and TM two proteins of transmembrane or gp41 are expressed. In addition, Pol expresses three proteins, PR (protease), reverse transcriptase (RT), and IN (integrase), and these proteins exhibit enzymatic activity.

Among the many proteins expressed above, the NC protein is a basic protein composed of 55 amino acids and has a molecular weight of about 6 kDa, comprising two zinc finger domains, which bind to the genomic RNA of the virus, Protect genomic RNA from nucleases. In addition, they specifically recognize viral genomic RNA, participate in its packaging, and also participate in RNA encapsidation and virion assembly. In particular, zinc finger domains are essential for the recognition of RNA, and basic residues are essential for RNA immobilization and virion assembly. Thus, studies have been conducted to neutralize HIV-1 by inhibiting the function of NC (Lener, D. et al., FEBS Letters., 361: 85-88, 1995; Dexter, TK et al., J. Virol., 70: 6607-6616, 1996; Tanchou, V. et al., J. Virol., 72: 4442-4447, 1998; Rong, L. et al., J. Virol., 72: 9353-9358 , 1998; Berthoux, L. et al., J. Virol., 71: 6973-6981, 1997; Berthoux, L. et al., J. Virol., 73: 10000-10009, 1999; Clever, J Let et al. , J. Virol., 74: 541-546, 2000; Cimarelli, A. et al., J. Virol., 74: 3046-3057, 2000). For example, Korean Patent Application No. 01-52594 discloses an RNA capable of binding to an NC protein, but RNA has a disadvantage in that it is difficult to work, and thus practically cannot be used for the treatment of HIV-1.

Therefore, a method for overcoming the above-described problems is being studied. One is to develop a protein or peptide that can inhibit the activity of HIV-1 by combining with NC protein, and the other is to target NC, not NC protein. It converts into psi sequences that bind to proteins. The psi sequence is a sequence consisting of 125 bases located at the 5 'end of the HIV genomic RNA. The psi sequence specifically binds to the NC protein to perform genome package of HIV.

Currently, the direction of the research is directed toward developing a protein capable of binding to NC protein, and no research results targeting psi sequences have been reported. However, in the replication of HIV, however, when targeting a relatively small amount of psi compared to the amount of NC protein expressed, peptides composed of fewer amino acids can be used to inhibit more effective HIV growth. Because it is expected to be, the research has been gradually progressed recently, but the situation is not yet achieved.

Therefore, the need to develop a peptide that can inhibit the proliferation of HIV in combination with the psi sequence of HIV is constantly emerging.

Therefore, the inventors of the present invention sought to develop a peptide capable of inhibiting the proliferation of HIV by binding to the psi sequence of HIV. As a result, using a phage display system, the present inventors consisted of 12 amino acids detected. It was confirmed that the peptide can specifically bind to the psi sequence, and completed the present invention.

After all, the main object of the present invention is to provide a peptide capable of inhibiting the proliferation of HIV-1 by binding to the psi sequence.

Another object of the present invention is to provide a gene encoding the electric peptide.

A peptide capable of inhibiting the proliferation of HIV-1 by binding to the psi sequence of the present invention is a peptide having an amino acid sequence of SYQWWWHSPQTL (SEQ ID NO: 1), and the electric peptide is a conventional method in the art from various genes encoding it. Can be expressed by.

The present inventors used a phage display system (PDS) to select peptides that can inhibit the proliferation of HIV-1 by binding to psi sequences. PDS expresses random peptides in gIII protein, a type of envelope protein of single strand filamentous bacteriophage M13, and selects peptides that bind strongly to specific target proteins, DNA or RNA. (biopanning).

Biopanning coats, blocks, washes, binds phage display-peptide library, washes, removes unbound phages, elutes bound phages, and amplifies eluted phages by coating, blocking, washing, and targeting specific target proteins, DNA or RNA on the plate. It contains a series of steps. When the electrical system is used, it is possible to quickly detect a nucleotide sequence encoding a ligand and screen a large amount of peptides in a short time, and thus has been usefully used in various fields.

The inventors constructed a plasmid for replicating the psi sequence to obtain a psi RNA sequence from the PDS sequence to select peptides that can inhibit the proliferation of HIV-1 by binding to the psi sequence using PDS. . Subsequently, as a result of performing PDS using the psi RNA nucleotide sequence obtained above, it was found that the peptide having the amino acid sequence was selected, and the peptide selected above inhibited the binding of psi RNA and NC. Therefore, since the peptide can be suppressed to proliferate human immunodeficiency virus by administering the peptide to cells infected with human immunodeficiency virus, it can be widely used in the development of a substance that can suppress the infection of HIV.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Performance of Phage Display System Using psi RNA

After amplifying a known psi sequence by PCR method, it was linked to obtain a tetramer psi gene, and inserted into the expression vector pUC18 to construct an expression vector, and then the electric expression vector was transferred to Escherichia coli BL21 (DE3). ) To obtain the transformants. The transformants were then cultured and mRNAs of tetrameric psi expressed therefrom were obtained.

The tetrameric psi RNA (2.1 μg) obtained above was activated with 0.2 M EDC (1-ethyl-3-dimethylaminopropyl-carbodiimide), and then reacted with 100 mM methylimidazole, followed by addition to coballink. , 5 hours at 50 ℃, was coated with psi RNA tetramer. The residue is then removed from cobalt, washed with TBST wash (TBS buffer + 0.05% Tween20), and then the blocking solution (50 mM Tris, 1 mM EDTA, 0.1% acetylated BSA, pH 7.5) is placed in coballink , 1 hour at room temperature, blocking was performed.

10 ¹⁰ PFU of phage was placed in psi RNA-coated cobaltlink, left at room temperature for 1 hour, then the solution was removed and washed 5 times with TBST (TBS buffer + 0.05% rween20) wash solution to retain the remaining unbound. Phage was removed. Subsequently, phage elution buffer (0.2M glycine, pH 2.2, 1 mg / ml BSA) was added to separate phages from RNA, and the isolated phages were added to E. coli ER2738 for infection, and then infected E. coli was cultured in a solid medium. The phage was amplified.

To determine the amount of electroinfected phage, titration was performed. That is, the phage and Escherichia coli ER2738 used in the above were prepared separately, and the phage were sequentially diluted in the order of 10%, 1%, 0.1%, 0.01% in TBS buffer solution (50 mM Tris, HCl pH 7.5, 150 mM NaCl). Then, 200 μl of E. coli ER2738, prepared separately, and 4 ml of Agarose-Top were mixed and poured into an LB solid medium to which IPTG and X-gal were added, followed by curing at 37 ° C. for one day. Thereafter, the expressed blue plaque was confirmed to determine an appropriate amount of phage dilution.

The electro-amplified phages were added to a culture medium in which E. coli ER2738 and LB liquid medium were mixed at 1: 100 (v / v) and shaken at 37 ° C. for 4 hours and 30 hours. Centrifugation gave supernatant.

PEG was added to the obtained supernatant to precipitate the phage, and this was obtained. The phage DNA extraction kit (M13 phage DNA isolation kit, Bioneer, Korea) was used to isolate the DNA of the phage, and confirmed their nucleotide sequence. As a result, it was found that the amino acid sequence of the peptide bound to psi RNA was SYQWWWHSPQTL (SEQ ID NO: 1).

Example 2 Effect Analysis of Peptides

The peptide of SEQ ID NO: 1 was synthesized using a peptide synthesizer (solid-phase peptide synthesizer, peptron, Korea), and gel shift assay was performed (see FIG. 1). Figure 1 is an electrophoresis picture showing the results of the gel shift assay to determine the binding of the peptide and psi, lane 1 is psi RNA + NC, lane 2 is psi RNA + NC + pep5, wherein , pep5 refers to a peptide having the amino acid sequence of SYQWWWHSPQTL (SEQ ID NO: 1) identified in Example 1 above. As shown in FIG. 1, when pep5 does not exist, psi RNA binds to NC and increases its molecular weight. However, when pep5 is present, it was found that psi RNA does not bind to NC. It was found to inhibit the binding between NCs.

As described and demonstrated in detail above, the present invention provides a gene encoding an electric peptide and a peptide specifically binding to the psi RNA sequence of HIV. A peptide capable of inhibiting the proliferation of HIV-1 by binding to the psi sequence of the present invention is a peptide having an amino acid sequence of SYQWWWHSPQTL. The peptide of the present invention binds to the psi RNA sequence, inhibits the binding between the psi RNA sequence and the NC protein, thereby inhibiting the proliferation of HIV, and thus may be widely used for the treatment of effective acquired immunodeficiency syndrome.

Figure 1 is an electrophoresis picture showing the results of performing a gel shift assay to determine the binding of the peptide and psi.

<110> MYUNG, Hee Joon YOU, Ji Chang <120> Peptide for Binding to psi RNA Sequence of HIV <160> 1 <170> KopatentIn 1.6 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> psi RNA binding peptide <400> 1 Ser Tyr Gln Trp Trp Trp His Ser Pro Gln Thr Leu 1 5 10

Claims (2)

A peptide that binds to the psi RNA of human immunodeficiency virus and has the amino acid sequence of SEQ ID NO: 1. Gene encoding the peptide of claim 1.